The goal of this research is to understand the mechanisms by which a protein folds spontaneously to a unique three-dimensional conformation. This research project will exploit the fact that all the information required for folding is contained in the amino acid sequence, by studying the folding behavior of proteins whose sequence has been altered by missense mutation. The system to be studied is a bacterial protein, the Alpha subunit of tryptophan synthase. A large number of strains of Escherichia coli with missense mutations in the Alpha gene region of the tryptophan operon have been isolated and the amino acid substitution in the Alpha subunit identified. In vitro equilibrium and kinetic studies of the reversible unfolding transition of wild-type and mutant proteins will monitor unfolding by changes in optical properties and by calorimetry. Site-directed mutagenesis will also be used to introduce amino acid replacements at new positions in the sequence. The results will be interpreted in terms of a folding model for the Alpha subunit, developed in our laboratory, which will allow quantitative comparison of the effect of the amino acid replacement. From this comparison, it will be possible to learn which amino acids play a key role in the folding process. These conclusions will then be combined with structural information from x-ray studies and predictive schemes in order to understand the effects of the mutation at a molecular level. Knowledge of the involvement of these key amino acids in stabilizing secondary and tertiary structures in the native, folded conformation will permit an assessment of those features that are critical to folding. This information will be useful in predicting the tertiary structure from amino acid sequence, in understanding the molecular basis of inherited diseases and in the design of new enzyme catalysts.